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| United States Patent |
5,714,343
|
|
Tuompo
, et al.
|
February 3, 1998
|
Method and kit for the detection of microorganisms
Abstract
A method for determining the presence of microorganisms in a liquid sample.
The liquid sample is first filtered through a filter having a pore size
which is small enough to prevent passage of microorganisms through the
filter but large enough to permit passage of any free reducing agents
present in the sample whereby microorganisms present in the sample are
retained on the filter. A chromogenic reagent having an oxidation
potential such that the reagent can be reduced by microbial dehydrogenase
and selected such that reduction of the chromogenic reagent yields a
visibly colored product is then passed through the filter, and the filter
is monitored for the formation of a visibly colored product indicative of
the presence of microorganisms in the sample. The method can be practiced
with the aid of a kit which includes a filter apparatus, a test solution
for visualization of microorganisms and optionally a solution of a
non-ionic alkyl glucoside-type detergent and a chaotropic agent for
visualization only gram-negative bacteria.
| Inventors:
|
Tuompo; Helena (Espoo, FI);
Glasin; Helja (Espoo, FI)
|
| Assignee:
|
Orion Corporation Ltd. (FI)
|
| Appl. No.:
|
450630 |
| Filed:
|
July 31, 1995 |
Foreign Application Priority Data
| Jan 24, 1991[FI] | 910358 |
| Jan 24, 1991[FI] | 910359 |
| Current U.S. Class: |
435/29; 435/4; 435/30; 435/34; 435/38; 435/39; 435/810 |
| Intern'l Class: |
C12M 001/12 |
| Field of Search: |
435/4,26,29,34,38,39,36,810,30,308.1
|
References Cited
U.S. Patent Documents
| 4724204 | Feb., 1988 | Steinbach et al. | 435/26.
|
| 5081017 | Jan., 1992 | Longoria | 435/34.
|
| 5366872 | Nov., 1994 | Hird et al. | 435/31.
|
| 5366873 | Nov., 1994 | Eden et al. | 435/34.
|
| 5420017 | May., 1995 | Tuompo et al. | 435/29.
|
Other References
An 82:178725 McKinnon et al. J. Appl. Bacteriol 51(2) 1981, pp. 363-368.
An 83:210725 Herson et al, AM Water Works Assoc J. 74(10) 1982 pp. 537-539.
An 84103166 Dutton et al, Applied and Environment Microbiology 46, (6)
1983, pp. 1263-1270.
|
Primary Examiner: Tran; Lien
Attorney, Agent or Firm: Brumbaugh, Graves, Donohue & Raymond
Parent Case Text
This application is a continuation of application Ser. No. 07/740,320,
filed on Aug. 5, 1991 now U.S. Pat. No. 5,420,017.
Claims
We claim:
1. A method for the detection of microorganisms in a liquid sample
comprising the steps of
(a) filtering the liquid sample through a filter having a pore size which
is small enough to prevent passage of microorganisms through the filter
but large enough to permit passage of any free reducing compounds present
in the sample, whereby microorganisms present in the sample are retained
on the filter;
(b) passing a test solution comprising a chromogenic reagent through the
filter having the retained microorganisms thereon, said chromogenic
reagent having an oxidation potential such that the reagent can be reduced
by microbial dehydrogenase and said chromogenic reagent being selected
such that reduction of the chromogenic reagent causes precipitation of the
chromogenic reagent on the filter to yield a colored product; and
(c) monitoring the filter for the formation of a colored product, wherein
the formation of a colored product is indicative of the presence of
microorganisms in the liquid sample.
2. A method according to claim 1 further comprising the step of
prefiltering the liquid sample through a prefilter having a pore size
which permits passage of microorganisms but retains mammalian cells and
particulate materials present in the liquid sample prior to step (a).
3. A method according to claim 1, further comprising the step of washing
the microorganism retained on the filter prior to addition of the test
solution.
4. A method according to claim 1, wherein the chromogenic reagent is a
tetrazolium salt.
5. A method according to claim 4, wherein the chromogenic reagent is
selected from the group consisting of triphenyltetrazolium chloride,
iodonitrotetrazolium, neotetrazolium chloride, blue tetrazolium and
nitroblue tetrazolium.
6. A method according to claim 1, wherein the chromogenic reagent contains
an electron transfer mediator effective to accelerate and amplify the
hydrogenation reaction selected from the group consisting of phenazine
methosulphate (PMS), menadione, meldola blue or methoxy phenazine
methosulphate.
7. A method according to claim 1, wherein the pore size of the filter is
from 0.75 to 1.2 microns.
8. A method according to claim 1, wherein the liquid sample is urine.
9. A test kit for the detection of microorganisms in a liquid sample
comprising, in packaged combination,
(a) a filter apparatus comprising a filter having a pore size which is
small enough to prevent passage of microorganisms through the filter but
large enough to permit passage of any free reducing agents present in the
sample whereby microorganisms present in the sample are retained on the
filter and means for drawing the liquid sample through the filter;
(b) a test solution comprising a chromogenic reagent, said chromogenic
reagent having an oxidation potential such that the reagent can be reduced
by microbial dehydrogenase and said chromogenic reagent being selected
such that reduction of the chromogenic reagent yields a colored product;
and
(c) a sterile wash solution comprising a buffer solution.
10. A test kit according to claim 9, wherein the filter apparatus further
comprises prefiltering means having a pore size which permits passage of
bacterial but retains mammalian cells and particulate materials present in
the liquid sample.
11. A test kit according to claim 9, wherein the means for drawing liquid
through the filter is a layer of adsorbent material disposed in contact
with the filter.
12. A test kit according to claim 9, wherein the pore size of the filter is
from 0.75 to 1.2 microns.
Description
BACKGROUND OF THE INVENTION
This application relates to a method and test kit for the detection of
microorganisms in a liquid sample. The invention relies upon the ability
of microbial dehydrogenase enzymes to reduce certain materials to produce
visibly colored and therefore detectable products and capitalizes on this
ability to produce simple and reliable kits to detect microorganisms such
as bacteria generally or just gram negative bacteria.
It has long been recognized that dehydrogenases in living cells can be
localized by histochemical methods within leucocytes and histological
tissue sections by replacing the natural hydrogen acceptor with a
reducible material, such as nitroblue tetrazolium as a ditetrazolium
chloride, having a colored, water insoluble reduction product. Michel, G.
et al. Methods in Enzymatic Analyses, Vol, 1, pp. 197-232 (1983). A
similar approach has been used to detect living bacteria by observing
whether a color change occurs upon the addition of a material such as
triphenyl tetrazolium chloride (TTC), methylthiazolyldiphenyl tetrazolium
bromide (MTT), iodonitrotetrazolium (INT) Or neotetrazolium chloride
(NTC), all of which turn red upon reduction, or blue tetrazolium (BT) or
nitroblue tetrazolium (NBT) which turn blue upon reduction. Histological
and Histochemical Methods, Theory and Practice, 2d. Ed. Chapter 16, p. 258
(1990).
The ability of living cells to reduce tetrazolium salts has formed the
basis of a research tool to investigate the effects of various materials
on bacterial respiration, i.e., oxygen uptake. In this regard, it has been
reported that anionic detergents such as Tergitol-7 selectively inhibit
the respiration of Gram-negative bacteria and consequently the reduction
of tetrazolium salts to colored products. Cationic detergents inhibit
respiration of both Gram-negative and Gram-positive bacteria, whereas
neutral, non-ionic detergents are reported to have no effect on
respiration. Baker et al., J. Exp. Med. 73, pp. 249-271 (1941).
Dehydrogenase activity appears to be inhibited as well. Dakay et al.,
Zentralblatt Bakt. Hyg. I. Abt. Orig. B. 174, pp. 121-124 (1981).
The differential effects of detergents on dehydrogenase activity has been
used as the basis for identifying bacterial types in liquid samples.
European Patent Application 107594. It is an object the present invention
to further exploit this ability of living bacteria and provide a method
and test kit for simple and rapid determination of total microorganisms
and Gram-negative bacteria.
SUMMARY OF THE INVENTION
In accordance with the present invention, the presence of microorganisms in
a liquid sample can be determined by a method comprising the steps of
(a) filtering the liquid sample through a filter having a pore size which
is small enough to prevent passage of microorganisms through the filter
but large enough to permit passage of any free reducing agents present in
the sample whereby microorganisms present in the sample are retained on
the filter;
(b) passing a test solution comprising a chromogenic reagent through the
filter having the retained microorganisms thereon, said chromogenic
reagent having an oxidation potential such that the reagent can be reduced
by microbial dehydrogenase and said chromogenic reagent being selected
such that reduction of the chromogenic reagent yields a visibly colored
product; and
(c) monitoring the filter for the formation of a visibly colored product,
wherein the formation of a visibly colored product is indicative of the
presence of microorganisms in the sample. This method can be practiced
with the aid of a kit which is also an aspect of the invention.
Either the method or the kit of the invention may be particularly adapted
for the detection of Gram-negative bacteria. In this case, a combination
of a non-ionic alkyl glucoside-type surfactant and a chaotropic agent is
added to the sample before filtration or included as a sterile wash
solution which is passed through the filter after the sample and before
the chromogenic reagent.
FIG. 1 shows an exploded view of a first apparatus useful in practicing the
present invention; and
FIG. 2 shows a second apparatus useful in practicing the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a highly effective means for detecting the
presence of microorganisms, particularly bacteria and more particularly
Gram-negative bacteria, directly from biological samples. The method is
particularly applicable for use with samples of urine, blood, milk add
water. The method can also be used on samples of process solutions, i.e.,
in the wood and pulp industry., the sugar industry or urban waste water,
and other liquid samples in which the presence of microorganisms is of
interest.
As used herein, the term "microorganism" refers to bacteria and yeasts.
In its simplest form, the method of the invention involves a three step
process: filtering the sample, adding the test solution, and observing the
result. The filtration Step is performed by passing the liquid sample
through a filter having a pore size which retains microorganisms on the
filter but allows any free reducing compounds which may be present to pass
through the filter. Examples of free reducing compounds that may be
present include soluble enzymes, ascorbic acid, pharmaceutical compounds
and low molecular weight sulfhydryl-containing molecules such as
glutathione. Suitable filters for this purpose are depth filters made up
of randomly stratified fibres, and having a pore size from 0.75 to about
1.2 .mu.m. Such matrix materials include, but are not intended to be
limited to, cotton, glass fiber, woven materials as cloth, nylon, or other
polymeric material. Bacteria, although being less than 1 .mu.m size are
entrapped on and into the filter by mechanical retention.
After filtration to separate microorganisms in the sample from any free
reducing compounds present in the sample, a test solution containing a
chromogenic reagent is added and drawn through the filter. The chromogenic
reagent can be essentially any material having an oxidation potential such
that it can be reduced by microbial dehydrogenase to produce a colored
product. Preferred reagents are those that are most rapidly reduced and
those that produce the most intensely colored products. Suitable
chromogenic reagents include tetrazolium salts, such as
triphenyltetrazolium chloride, iodonitratetrazolium, neotetrazolium
chloride, blue tetrazolium and nitroblue tetrazolium; and other materials
such as methylene blue, dichloroindophenol and resazurin.
The test solution may also contain an electron transfer mediator effective
to accelerate and amplify the hydrogenation reaction. Suitable mediators
include phenazine methosulphate (PMS), menadione, meldola blue (C.I.
51175, Basic Blue 6) and methoxy phenazine methosulfate.
Passing the test solution through the filter causes microorganisms on the
filter to be "dyed," making them immediately detectable. For example, a
light yellow solution of NBT when reduced forms a visible, blue, formazane
precipitate with considerable rapidity on the filter if living
microorganisms are present. Indeed, NBT is a preferred chromogenic reagent
for use in the invention as the blue color of reduced NBT is formed
considerably more rapidly than the red of TTC and is easier to distinguish
from the red color of samples containing hemoglobin.
NBT and MTT are equally rapid from the point of view of color formation,
but the advantage of NBT in comparison with MTT is that the color can be
deposited on a restricted area of the filter because it precipitates at
the point where the dehydrogenases are situated around the bacteria.
Excess NBT does not dissolve the blue formazane. Using NBT bacteria can be
identified with rapidity at room temperature by visual inspection and
without any separate measuring equipment. MTT is water-soluble and thus is
not retained on the fiber but it can be used in spectrophotometric
methods.
The amount of chromogenic reagent present in the test solution is not
critical, e.g. NBT from 0.2 mg/ml to 2.0 mg/ml are suitable and depends on
the speed with which the reagent is reduced, (which controls the amount of
product produced), NBT and MTT being rapid and leading to high extinction
coefficient of the reduced product (i.e. the intensity of the color
produced) and to the level of sensitivity required. The speed of the
reduction can be varied by the addition of an accelerator. In general,
test solutions containing from 0.2 mg/ml to 2.0 mg/ml of chromogenic
reagent and 0.1 to 10 g/l of an accelerator such as PMS are suitable.
The basic method of the invention can be modified in several ways to adapt
to certain special circumstances. For example, for samples which may
contain leucocytes or other cells which can themselves reduce the
chromogenic reagent but which are not of interest in the assay, a
prefiltration step may be added. Suitable prefilters have a pore size of
10 to 20 .mu.m which retains leucocytes and other mammalian cells but,
permits passage of yeast and bacteria.
A washing step may also be added between the filtration step and the
addition of the test solution. This washing, can be performed with buffer
to further remove erroneous results caused by free-reducing compounds.
If Gram-negative bacteria are of special interest, the sample itself or the
washing buffer can be modified inhibit the dehydrogenase enzymes of all
but Gram-negative bacteria. This is done using a non-ionic alkyl
glucoside-type detergent, for example octyl glucoside or octyl maltoside,
and a chaotropic compound such as guanidine or urea. Other non-ionic
detergents such as Triton X-100 and Tween 80, which are polyxyethelyne
compounds, have no effects under the same conditions, as was reported much
earlier by Dakay et al. (1981).
If present in the sample solution or in the wash buffer, the non-ionic
alkyl glucoside detergent is suitably used at a concentration of from 8
mg/ml to 12 mg/ml.
Chaotropic compounds are suitably used at concentrations of from 0.5 to
1.0M in the sample solution or in the wash buffer.
The method of the invention can be practiced using any filtration device,
including laboratory devices in which vacuum is used to draw liquid
through the filter. For use in a kit, however, a filter apparatus in which
an absorbent material is disposed on one side of the filter to draw liquid
through the filter is more convenient. Two apparatus of this type are
shown in FIGS. 1 and 2.
FIG. 1 presents an exploded view of an apparatus in which a liquid sample
e.g. urine is pipetted onto the prefilter 1 if the sample is particulate
or if it contains large numbers of leucocytes (in other cases
prefiltration is not necessary). In the collection chamber 2 the microbes
in the sample are trapped on the filter 3. The sample is prebuffered or
washed with a buffer solution after filtration the sample to detect all
microbes.
To detect only the gram-negative bacteria the detergents are added to the
sample with the buffer solution or to the wash solution.
After filtration the bacteria are visualized by detecting their own
dehydrogenases which form blue formazane precipitate on the filter. Below
the filter 3 is an adsorbent material 4, which absorbs the sample liquid.
All the parts of the apparatus takes the form of a small box.
The apparatus depicted in FIG. 2 includes a prefilter 1 for removing
particles from the sample, a filter 3 on which bacteria are trapped and an
adsorbent material 4 which may be attached e.g. using two-sided tape to
the base 5. The buffered sample is adsorbed firstly to the prefilter when
dipping the edge of filter 1 to the buffered sample solution. The
particulate matter or leucocytes in the sample are retained on the filter
1 and bacteria present in the sample will move to the interface between
prefilter 1 and filter 3, and the liquid part of the sample will penetrate
filter 3 and possibly to the adsorbent material 4. The bacteria can be
demonstrated by dipping the edge of prefilter 1 to the chromogenic
solution and blue formazane due to bacterial own dehydrogenases is
produced around the bacteria. If only gram-negative bacteria are
visualized the detergents maybe added to the buffered sample and to the
chromogenic solution.
In the following examples, The test bacteria were types isolated from
urinary tract infections, either ATCC type strains or clinical strains
obtained from the Department of Serobiology of the University of Helsinki.
The Gram-negative bacteria were Escherichia coli (ATCC 25922 and several
clinical strains): Klebsiella aerogenes (ATCC 13833), Proteus mirabilis,
Pseudomonas aeruginosa and Enterobacter aerogenes (ATCC 13048).
Gram-positive strains included Staphylococcus aureus (ATCC 25923),
Staphylococcus saprophyticus, Staphylococcus epidermidis, Streptococcus
agalactiae, Streptococcus faecium (ATCC 9790), .beta.-hemolytic
Streptococcus, group B, Enterococcus sp. and Corynebacterium sp. The yeast
strains were Candida albicans ATCC 28367 and 36802. The bacteria were
cultivated overnight in BHI broth (Brain-Heart Infusion, Difco, Detroit)
at 37.degree. C. and the bacterial count in the broth was adjusted to
about 10.sup.8 CFU/ml (colony forming units/ml) by dilution with sterile
0.9% sodium chloride solution to an optical density of 0.400 at 650 mm
(Perkin Elmer Spectrophotometer, Norwalk). The bacterial population was
checked by dilution and plating on blood agar, The yeast strains were
cultivated for 2 days at 37.degree. C. on Sabouraud agar plates and
suspensions of 10.sup.8 CFU/ml were made by transferring microbial mass to
sterile salt solution to an optical density of 0.400 as described above.
In addition to these samples, 100 urine samples were collected. The
bacterial contents of these samples were assayed by bacterial cultivation
(Uricult.RTM., Orion Diagnostica). Leucocytes possibly present in the
samples were collected on a leucocyte filter (Pall Biosupport Company,
Glen Cove).
In the assay 100 .mu.l of bacterial suspension was added to 100 .mu.l 0.2M
Tris-HCL buffer, pH 6.5, 7.2 or 8.5 (Sigma, St. Louis) containing 1.0%
glucose (BDH, Poole) or fructose (BDH, Poole), acetate (Merck, Darmstadt)
or glutamate (Merck, Darmstadt). For some assays the mixture also
contained varying amounts of octyl glucoside (Sigma, St. Louis) or
guanidine hydrochloride (BDH,Poole) and was incubated for different times
on a 96-place well plate. The sample was pipetted to a filtering
apparatus, on which the bacteria were collected in an area of about 3 mm
diameter on the surface of the filter (Schleicher and Schuell glass fiber
filter No. 8), or the filter (leucocyte filter, Pall BioSupport Company,
Glen Cove) was dipped into the sample and the bacteria passed through it
and transferred to the filter interface where another filter (Schleicher
and Schuell No. 8) was positioned. The bacteria were detected by adding
100 .mu.l NBT-solution (1 mg/ml, Sigma, St. Louis) containing 10 .mu.l PMS
(1 mg/ml, Sigma, St. Louis) to the filter.
Color formation was estimated visually after different times according to
the scale:
0=colorless
1=weak color
2=clearly detectable color
3=strongly colored
4=darkly colored
5=very darkly colored
EXAMPLE 2
Detection of micro-organisms at different concentrations was carried out as
described in Example 1. The micro-organisms were incubated for a short
time on a shaker and then for 30 seconds in 0.2M Tris-HCL buffer
containing 10% glucose, at different pH values. The suspension was then
transferred to a filter, to which a solution of NBT-PMS was added and the
blue color due to production of formazane was read after 30 minutes. The
results are summarized in Table 1.
TABLE 1
______________________________________
Test bacteria, CFU/ml
pH
›Bact./ml! 6.5 7.2 8.5
______________________________________
E. Coli (ATCC 25922)
10.sup.7
2 3 3
10.sup.6
2 2 2
10.sup.5
0 0 0
E. Coli a 10.sup.7
4 4 3
10.sup.6
2 2 2
10.sup.5
0 0 0
E. Coli b 10.sup.7
4 4 4
10.sup.6
0 2 3
10.sup.5
0 0 0
E. Coli 2956 10.sup.7
2 3 1
10.sup.6
1 1 1
10.sup.5
0 0 0
E. Coli 3338 10.sup.7
4 5 5
10.sup.6
2 2 2
10.sup.5
0 0 0
E. Coli 110515 10.sup.7
2 3 3
10.sup.6
2 2 2
10.sup.5
0 0 0
Klebsiella aerogenes
10.sup.7
5 5 5
(ATCC 13883) 10.sup.6
4 4 5
10.sup.5
2 3 3
Enterobacter aerogenes
10.sup.7
5 5 5
(ATCC 13883) 10.sup.6
4 5 5
10.sup.5
3 3 3
Proteus mirabilis
10.sup.7
5 5 5
10.sup.6
3 3 4
10.sup.5
1 1 2
Pseudomonas aeruginosa
10.sup.7
4 4 4
10.sup.6
2 2 2
10.sup.5
1 2 2
Enterococcus sp. 10.sup.7
5 5 5
10.sup.6
3 3 3
10.sup.5
2 2 2
Streptococcus agalactia
10.sup.7
5 5 5
10.sup.6
3 2 3
10.sup.5
1 2 1
Streptococcus 10.sup.7
5 5 5
beta-hemol.B 10.sup.6
3 4 4
10.sup.5
1 2 2
Streptococcus 10.sup.7
4 4 4
faecium (ATCC 9790)
10.sup.6
3 3 3
10.sup.5
1 2 2
Staphylococcus 10.sup.7
3 5 5
aureus 10.sup.6
2 3 3
10.sup.5
0 1 2
Staphylococcus 10.sup.7
4 4 4
epidermidis 10.sup.6
2 2 3
10.sup.5
0 1 2
______________________________________
EXAMPLE 3
Different clinical strains of Escherichia coli were detected on the filter
at different concentrations. The bacteria were incubated for 30 seconds in
0.2M Tris-HCL buffer, pH 7.2 containing 1.0% glucose and transferred to a
filter to which NBT-PMS solution was added and the intensity of the blue
color of formazane was estimated after 30 minutes, The results are shown
in Table 2.
TABLE 2
______________________________________
CFU/ml
Bacterial strain
10.sup.9 10.sup.8
10.sup.7
10.sup.6
10.sup.5
______________________________________
E. coli a 5 5 4 2 0
E. coli b 5 4 3 2 0
E. coli 2956
4 4 3 1 0
E. coli 3338
5 5 5 2 0
E. coli 110515
4 4 3 0 0
______________________________________
The ability of guanidine to prevent formazane production by a Gram-positive
bacterium, Staphylococcus saprophyticus, versus the effect on the
Gram-negative E. coli was determined. The test was performed as in Example
2 and with the addition of 0.5M guanidine.
The results are shown in Table 3. As can be seen from Table 3, guanidine at
this concentration essentially eliminated the formazane producing activity
of the Gram-positive organism.
TABLE 3
______________________________________
No Additives
Guanidine
Test bacteria,CFU/ml
pH
Bact./ml 6.5 7.2 8.5 6.5 7.2 8.5
______________________________________
E. Coli 10.sup.7
2 3 3 2 3 3
(ATCC 25922)
10.sup.6
2 2 2 2 2 2
10.sup.5
0 0 0 0 0 0
Staphylococcus
10.sup.7
4 4 4 0 0 1
saprophyticus
10.sup.6
0 2 3 0 0 0
10.sup.5
0 0 0 0 0 0
______________________________________
Prevention of formazane production by different bacteria with 0.5M
guanidine at pH 7.2, in the presence of different substrates (1% glucose;
1% fructose; 1% glutamate; 1% acetate) was studied. In other respects the
test was carried out as in the case of Example 1. The results shown in
Table 4, indicated that contrary to the initial expectation raised by
Example 4, guanidine alone cannot generally be used to selectively inhibit
formazane production by Gram-positive organisms.
TABLE 4
______________________________________
Test bacteria, CFU/ml
gluc. fruct. glutam acet.
______________________________________
GRAM - 10.sup.7
5 5 4 3
Escherichia coli a
10.sup.6
4 4 3 2
GRAM + Streptococcus,
10.sup.7
5 5 -- 5
Group B, 102 10.sup.6
2 2 -- 1
GRAM - 10.sup.7
5 5 -- 5
Klebsiella aerogenes
10.sup.6
4 4 -- 0
(ATCC 13883)
GRAM + Streptococcus,
10.sup.7
4 4 -- 2
faecium (ATCC 9790)
10.sup.6
0 0 -- 0
GRAM - 10.sup.7
5 5 -- 5
Enterobacter sp.
10.sup.6
4 4 -- 3
GRAM + Staphylococcus
10.sup.7
3 3 -- 3
epidermidis 10.sup.6
0 0 -- 0
GRAM - 10.sup.7
5 5 -- 5
Proteus mirabilis
10.sup.6
3 3 -- 3
GRAM + 10.sup.7
5 5 -- 2
Enterococcus, sp.
10.sup.6
0 0 -- 0
GRAM - Pseudomonas
10.sup.7
0 0 3 0
aeruginosa 10.sup.6
0 0 2 0
GRAM + 10.sup.7
5 5 3 5
Staphylococcus
10.sup.6
1 1 3 2
aureus
GRAM + 10.sup.7
3 3 0 2
Staphylococcus
10.sup.6
0 0 0 0
saprophyticus
______________________________________
EXAMPLE 6
Prevention of formazane production by different bacteria with octyl
glucoside at different concentrations was studied. In other respects the
test was carried out as in the case of Example 1 in the presence of 1%
glucose. The bacterial concentration was 10.sup.7 CFU/ml. for all
experiments. The results are shown in Table 5.
TABLE 5
______________________________________
Octylglucoside Concentration
(g/l)
4 6 8 10
______________________________________
GRAM -
Escherichia coli a 4 5 4 4
Klebsiella aerogenes
5 5 5 5
Enterobacter aerogenes
5 5 5 3
Proteus mirabilis 5 2 2 2
Pseudomonas aeruginosa
0 0 0 0
GRAM +
Staphylococcus aureus
5 0 0 0
Enterococcus sp. 4 0 0 0
Staphylococcus epidermidis
3 3 0 0
Staphylococcus saprophyticus
3 2 2 2
Streptococcus agalactiae
4 0 0 0
Streptococcus faecium
3 0 0 0
Streptococcus sp. B
5 0 0 0
______________________________________
EXAMPLE 7
Prevention of formazane production by different bacteria using 0.5M
guanidine with and without 10 g/l octyl glucoside in the presence of 1%
glucose. In other respects the test was performed as in the case of
Example 1. The bacterial concentration was 10.sup.7 CFU/ml for each
experiment. As shown in Table 6, the combination of chaotropic agent and
the nonionic glucosede surfactant provides substantially complete
specificity between Gram-positive and Gram-negative bacteria, and also
pseudomonas becomes usable, whereas the yeasts are not stained.
TABLE 6
______________________________________
octyl octyl
glucoside
glucoside
added not added
______________________________________
GRAM -
Escherichia coli a 4 5
Klebsiella aerogenes
4 5
Enterobacter aerogenes
4 5
Proteus mirabilis 5 5
Pseudomonas aeruginosa
4 0
GRAM +
Staphylococcus aureus
0 5
Enterococcus sp. 0 5
Staphylococcus epidermidis
0 4
Staphylococcus saprophyticus
0 4
Streptococcus agalactiae
0 5
Streptococcus faecium
0 4
Streptococcus sp. B
1 5
Corynebacterium sp.
0 5
Candida albicans 0 4
Candida albicans B 0 3
______________________________________
EXAMPLE 8
100 urine samples were analyzed with Uricult.RTM. and with the method
described in Example 2, in which bacteria were detected with the aid of
formazane. A positive result (+) of the Uricult.RTM. test indicates that
the sample contains at least 10.sup.5 bacteria/ml. Bacterial counts below
10.sup.5 CFU/ml give a negative result (-). A positive result with
formazane was recorded when the color intensity was 2 or greater and a
negative result was indicated by a color intensity of 0 or 1. The results
of this comparison, shown in Table 7, indicate that tests results with the
claims method are of comparable reliability of the commercial product.
TABLE 7
______________________________________
formation of formazane
Standard Test Type
Result + -
______________________________________
Total bacterial count,
+ 21 0
Uricult - 0 79
GRAM - reaction,
+ 12 1
- 0 87
GRAM + reaction,
+ 6 2
- 0 92
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